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Airborne microorganisms

The
Microbial World:Airborne Microorganisms

Produced
by Jim Deacon
Institute of Cell and Molecular Biology, The
University of Edinburgh

Airborne microorganisms

Airborne particles are a
major cause of respiratory ailments of humans, causing
allergies, asthma, and pathogenic infections of the
respiratory tract. Airborne fungal spores are also
important agents of plant disease, and the means for
dissemination of many common saprotrophic (saprophytic)
fungi.

From slide
collection, Department of Medical Microbiology, Edinburgh
University

During a sneeze, millions
of tiny droplets of water and mucus are expelled at about
200 miles per hour (100 metres per second). The droplets
initially are about 10-100 micrometres diameter, but they
dry rapidly to droplet nuclei of 1-4
micrometres, containing virus particles or bacteria. This
is a major means of transmission of several diseases of
humans, shown in the table below.

Some important diseases of humans
transmitted from person to person by inhaled airborne
particles

Virus diseases
(virus type in brackets)

Bacterial diseases
(bacterial name in brackets)

Chickenpox (Varicella)

Whooping cough (Bordetella pertussis)

Flu (Influenza)

Meningitis (Neisseria species)

Measles
(Rubeola)

Diphtheria
(Corynebacterium diphtheriae)

German
measles (Rubella)

Pneumonia
(Mycoplasma pneumoniae, Streptococcus species)

Mumps
(Mumps)

Tuberculosis
(Mycobacterium tuberculosis)

Smallpox
(Variola)

Several other diseases, below, are acquired by inhaling
particles from environmental sources, not directly from
an infected person.

Disease

Source

Psittacosis (Chlamydia
psittaci)

Dried, powdery droppings from infected birds
(parrots, pigeons, etc.)

Legionnaire's disease (Legionella
pneumophila)

Droplets from air-conditioning systems,
water storage tanks, etc., where the bacterium grows.

Psittacosis
is a serious disease acquired by handling birds or by
inhaling dust from bird faeces. It is caused by the
bacterium Chlamydia psittaci, an obligate
intracellular parasite. After entering the respiratory
tract, the cells are transported to the liver and spleen,
multiply there and then invade the lungs, causing
inflammation, haemorrhage and pneumonia.

Legionnaire's
disease is a fairly common form of pneumonia in
older or immunocompromised people. It is seldom
transmitted directly from person to person. The bacterium
is an aquatic rod-shaped species with a temperature
optimum of about 36oC, and is a common
inhabitant of warm-water systems in buildings. Infection
occurs when people inhale aerosol droplets containing the
bacteria.

Extrinsic allergic
alveolitis is a serious hypersensitive response,
usually associated with repeated exposure to airborne
spores in the work environment. A classic example is the
condition termed farmer's lung, caused
by exposure to spores of thermophilic actinomycetes.

Aspergillosis,
Histoplasmosis and Coccidioidomycosis
are examples of serious fungal infections of humans,
initiated by spores deposited in the alveoli. They can be
life-threatening diseases of immunocompromised people,
when the fungi disseminate from the lungs to major organs
of the body. However, in all cases the infection of
humans is incidental to the fungus, playing no part in
its normal biology. These are fungi that grow naturally
as decomposer organisms in soil, bird faeces or other
organic substrates.

Aspergillus
fumigatus. (A) Typical sporing heads of the
fungus in laboratory culture. Spores are produced from
phialides that arise from the upper part of a club-shaped
swelling (vesicle) of an erect hypha (the
sporangiophore). [See Thermophilic microorganisms]. (B) Microscopic section of lung tissue,
stained to show hyphae of Aspergillus in an air
sac. Such a ball of hyphae growing saprotrophically in
the lung is termed an aspergilloma.

Air sampling is used routinely to
monitor the populations of airborne particles, and to
inform the public about air quality and pollen/spore
counts through public broadcasting (weather reports,
etc.). It is used by major hospitals to monitor the
populations of specific allergenic particles (fungal
spores, etc.), so that the causes of patients' allergies
can be determined. And it is used in crop pathology for
disease-forecasting, so that growers can apply fungicides
as and when required.

Here we will consider
three major types of sampling device for detecting fungal
spore loads in air:

the rotorod sampler

the Burkard sampler

the Anderson sampler.

The Rotorod spore
sampler

The rotorod
sampler (FigureC
below) is a cheap, simple and portable air sampler. It
consists of a U-shaped metal rod attached by a spindle to
a battery-powered electric motor. The motor causes the
upright arms of the metal rod to rotate at high speed. To
use the sampler, the upright arms are covered with narrow
strips of sticky tape, so that any spores in the air will
impact onto the tapes. Then the tapes are removed and
examined microscopically to identify the spores and other
particles such as pollen grains in the air. Some examples
are shown in Figures D and E.

Figure C: Rotorod sampler. Figures D, E:
Pieces of sticky tape on which spores and other particles
were impacted. The identifiable particles include: in D,
an ascospore (as), a hyaline
(colourless) fungal spore (h) and a
conidium of the common leaf-surface fungus Cladosporium
(c); in E,
multicellular conidia (resembling snowshoes) of a common
leaf-surface fungus Alternaria (a),
conidia (c) and hyphal fragments (ch)
of Cladosporium, and a large hyaline spore of a
powdery mildew fungus (m).

This type of sampler is
most effective for trapping relatively large particles
(upwards of about 7 micrometres) such as the larger
fungal spores and pollen grains. The reason for this is
shown in Figure F below.

When air is travelling
towards an object such as a narrow cylinder, or
vice-versa, it is deflected around the object. Any
particles in the air will tend to continue along their
original trajectory, but their ability to do this (and to
impact onto the object) is governed by their momentum(defined as mass x velocity).
At any given air speed, the heavier particles are most
likely to impact (a in the diagram)
whereas smaller (lighter) particles are likely to be
deflected round the object. Very high air speeds would be
required to impact the smaller particles (b
in the diagram), and such air speeds are seldom found in
natural conditions.

In practice, therefore,
fungal spores and other airborne particles can be grouped
into two broad categories - those that can impact on
surfaces (impactors), and those that are
smaller and are only removed from the air by
sedimentation in prolonged calm conditions or that are
removed from the air by rain.

One of the advantages of
the rotorod sampler is that it can be used to precisely
locate a source of spores of a particular fungus. The
famous aerobiologist, PH Gregory, did this in the 1950s
by placing rotorod samplers at different positions in a
field and "homing in" on a source of spores of
the fungus Pithomyces chartarum, which causes a
condition known as facial eczema of sheep.

Many important pathogens
of crop plants have large spores that impact readily onto
plant surfaces to initiate infection. Examples include
powdery mildew of wheat, Erysiphe graminis (Figure
G, with spores about 30 micrometres long) and
loose smut of wheat, Ustilago tritici (Figures
H, I). This smut disease is characterised by a
mass of black spores where the grain would normally be
produced. These spores are 8-10 micrometres diameter.
(See Biotrophic pathogens).

The Burkard spore sampler acts on
the same principle as the rotorod sampler, but is used to
give a continuous record of particles in the air over a
period of 24 hours or up to 7 days. The apparatus (Figures
J, K) consists of an air-sealed drum that
contains a clockwork rotating disc (arrowhead in Fig. K)
which makes a single revolution in 7 days. The surface of
this disc is covered with adhesive tape, to trap spores
that impact onto it. When the apparatus is assembled, air
is sucked into the drum at high speed through a slit
orifice (arrowhead in Fig J) by means of a motor at the
base of the apparatus. Any particles in the air impact
onto the sticky tape near the slit orifice, giving a
record of the particles in the atmosphere at a specific
time of day. At the end of a 7-day run, the tape is
removed, cut into sections representing hourly or daily
periods, then examined microscopically.

In this way, it is
possible to distinguish clearly between night-released
and day-released spores or other particles, and also to
relate the types of particle to different weather
conditions (e.g. humid or dry periods) while the
apparatus was running. The Burkard spore trap is commonly
used for continuous monitoring of spore or pollen loads
in the air. For example, these traps are commonly sited
on hospital roofs, meteorological stations, and other
public buildings, and provide public information through
TV and radio broadcasts.

The principle is exactly
the same as in the rotorod sampler because the trapping
of particles is based on impaction. The limitations also
are the same: only the larger particles with sufficient
mass will impact on the tapes at the air speeds generated
by this type of sampler.

Figures
J-K. The Burkard spore
sampling device, shown in assembled form (J) and during
preparation (K).

The
Anderson sampler

The Anderson sampler (Figure
L) is an ingenious device for selectively
trapping different sizes of particles according to their
size (momentum). This sampler consists of a stack of 8
metal sections that fit together with ring seals to form
an air-tight cylinder. Each metal section has a
perforated base (see Figure N), and the
number of perforations is the same in each section, but
the size of these perforations is progressively reduced
from the top of the column to the bottom. To use this
sampler, open agar plates are placed between each metal
section, resting on three studs (shown as arrowheads in
Figure N).

Figures
L, M. The Anderson air sampler, shown in
a laboratory (L) and mounted with a
wind-vane in a field site (M).

When fully assembled (with
an open agar plate between each unit) an electric motor
sucks air from the bottom of the unit, causing
spore-laden air to enter at the top (arrowhead in Figure L)
and to pass down through the cylinder. The path taken by
this air is shown in Figure O, below.

Air sucked in at the top of the
column travels at
relatively low speed towards the first agar plate, and so
only the largest particles impact onto the agar surface.
The air then travels round the edge of the agar plate and
through the perforations to the second agar plate, and so
on. As this process continues down the stack, the same
volume of air is forced to travel through successively
smaller perforations, and so the air speed is
progressively increased. The progressively increased air
speed lower down the column raises the momentum of the
air-borne particles, so that even the very smallest
particles (less than 3 micrometres diameter) can impact
onto the lower agar plates.

When the sampler has run
for 5-15 minutes or more, the metal plates are separated
and the Petri dishes are removed for incubation to
identify the colonies that develop. Figures P-R(below)
show some examples of agar plates from an Anderson
sampler. In this case the air sample contained spores
from mouldy hay, and the agar plates were incubated at 37oC.

Figure
P: An agar plate from the bottom level
of an Anderson sampler. The colonies are of thermophilic
actinomycetes (Micropolyspora faeni or Thermoactinomyces
vulgaris) that are common causes of Farmer's
lung disease (extrinsic allergic alveolitus).
Actinomycete spores are very small (1-2 micrometres) so
they commonly enter the lungs. They form dense,
slow-growing colonies on agar, and the pattern of
colonies seen on this agar plate reflects the pattern of
the perforations through which the air had passed. This
Figure also shows how a divided (three-sectored) Petri
dish can be filled with different agar media to detect
different types of organism in the air. Figures Q
and R show agar plates from the
midlle part of the Anderson sampler, where several
species of Aspergillus and Penicillium
have developed from spores about 3-5 micrometres
diameter.

One of the interesting
features of the Anderson sampler is that it mimics the
deposition of spores (or other ariborne particles) in the
human respiratory tract (see Figure O).
For example, relatively large fungal spores and pollen
grains tend to be trapped on the mucus-covered hairs of
our nostrils, where they can cause "hay fever"
symptoms in sensitised individuals. Smaller particles are
not trapped in the nostrils but instead are carried down
into the bronchioles and alveoli. Here the air speed is
very low, because the successive branching of the
respiratory tract has reduced the air speed to a minimum.
But spores of about 2-4 micrometres diameter can settle
onto the mucosal surfaces of the alveoli. Some of these
spores are important in initiating infections of the
lungs. However, it is important to note
that the underlying mechanisms of spore deposition in the
Anderson sampler are entirely different from those in the
human respiratory tract - the Anderson sampler traps
spores by impaction, whereas spores are
deposited in the human respiratory tract mainly by
sedimentation.

The
human respiratory tract as an air-sampling device

The respiratory tract is highly
effective in trapping airborne particles, with sometimes
serious consequences for health. The mechanisms involved
depend on particle size.

Large particles
(about 10 micrometres) have sufficient mass to impact
onto surfaces, even at low air speeds. They break
free from the air as it flows around obstacles.
During normal breathing, the airflow in the nose
and trachea is about 100 cm per second -
sufficient for pollen grains and larger fungal
spores (Alternaria, etc.) to be retained on
the mucosa, where they can cause typical
hay fever symptoms like rhinitis and
asthma.

As we have seen, these are the types of particle
- the "impactors" - detected by the
rotorod and Burkard samplers, and also found on
the top plates of the Anderson sampler.

Smaller particles do
not impact at these air speeds, and the air speed
decreases as the repiratory system branches
further down. So all the particles of 5
micrometres or less are carried deep into the
lungs. There they can settle out by sedimentation
in the brief periods when the air is calm between
successive breaths. Particles of 2-4 micrometres
are optimal for alveolar deposition, and this
range includes the spores of many Aspergillus
and Penicillium species.

This is how some of the serious fungal infections
of humans are initiated - aspergillomas, Histoplasmosis,
Coccidioidomycosis, etc.

Even smaller
particles, such as the spores of actinomycetes (about 1 micrometre) are
less efficient at being deposited in the alveoli,
but repeated exposure to spore clouds by
agricultural workers can lead to sensitisation
and extrinsic allergic alveolitis
(Farmer's lung, etc.)

Very small particles,
less than about 0.5 micrometres, do not impact
but are moved by diffusion (Brownian
motion) which brings them randomly into
contact with surfaces in the lungs. This is true
of the fine dusts that cause many occupational
diseases.

Where do the
bacterial and viral pathogens fit into this scheme?

The nasopharyngial
infections by viruses are associated with large sneeze
droplets which impact in the upper airways. Most
bacterial diseases also are initiated in the upper
airways, when bacteria are carried in large droplets or
on "rafts" of skin that impact onto the mucosa.
However, infections by Mycobacterium (tuberculosis)
and Bacillus anthracis (anthrax)
are initiated in the lungs. These are highly virulent
pathogens, and even single cells or spores (about 3
micrometres for Bacillus) can initiate
infections after deposition in the alveoli.